Peptide-based technologies have facilitated the elucidation of disease mechanisms and serve as novel and effective therapeutics. An emerging theme in biotechnology is to use peptide variants to disrupt protein-protein interactions because compared to small molecules they have a larger surface area for binding, can recognize targets with higher specificity/affinity, and can be generated in weeks by phage display. For this and other reasons, for many years researchers in the field of bioconjugate chemistry have needed well-defined ligation strategies that can be used for the at-will modification of biomolecules. Efficient bioconjugation strategies generally involve high levels of functional group tolerance, compatibility with water and other solvents, and efficient conversions (e.g., fast reaction times and high yields).
Selenocysteine (Sec or U), discovered by Stadtman in 1974, is the 21st proteinogenic amino acid. Selenocysteine is a structural analogue to cysteine with a selenol in place of the thiol. There are several other notable differences between cysteine and selenocysteine. The greater acidity of selenocysteine (pKa=5.47) versus cysteine (pKa=8.14) causes it to be deprotonated under physiological pH and its lower reduction potential makes it an integral part of antioxidant proteins. Selenocysteine is essential for enzymatic activity in enzymes including glutathione peroxidases, iodothyronine deiodinases, formate dehydrogenases, and methionine-R-sulfoxide reductase. Mutation of the catalytic selenocysteine to cysteine in the aforementioned enzymes results in a decrease in activity of >100 fold.
The inherent nucleophilicity of selenols makes selenocysteine an appealing handle for chemoselective bioconjugation in peptides and proteins. Nevertheless, reports on bioconjugation with this amino acid have been sparse due to several challenges associated with its functionalization. Namely, a selenol is easily oxidized to the diselenide or seleninic acid. In addition, because selenium is highly polarizable, it can be eliminated to generate dehydroalanine. Reports of selenocysteine functionalization have paralleled methods to modify cysteine and relied upon alkylation and maleimide conjugate addition with the selenol group (FIG. 1, Panel (1)). In contrast to common cysteine conjugation methods, a reducing agent [i.e., tris(2-carboxyethyl)phosphine (TCEP)] and exclusion of oxygen are needed to generate the selenol in situ prior to reaction with the electrophile.
There exists a need for development of a direct, robust, and selective method for the functionalization of selenocysteine that overcomes these limitations and allow for site-selective modification of unprotected peptides.